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Edman Degradation Sample Preparation Protocols : Deblocking by pyroglutamdase

사람의 항체 및 천연의 단백질 중 가장 잘 알려진 blocking 형태인 pyroglutamate 의 제거는 다음으로 이어지는 아미노산 서열의 정확한 결정을 위하여 deblocking 과정 중 중요한 전처리 방법을 소개합니다.
Protocol for Deblocking Proteins with pfu Pyroglutamate Aminopeptidase (PGAP)
I. On Membrane.
1. Place PVDF sample in 0.5 ml Eppendorf tube. Wet membrane with 1 µl methanol.
2. BLOCK. Add 200 µl 0.5% polyvinyl pyrrolidone (PVP-360) in 0.1% Acetic acid. Shake for 20 minute at room temperature. Discard supernatant.
3. Rinse. Wash 3 times with 200 µl milli-Q water. Transfer PVDF to a 0.2 ml PCR reaction tube.
4. DIGEST. Add 30 µl of freshly made 1X PGAP buffer consisting of 50 mM Sodium Phosphate, 10 mM DTT, 1 mM EDTA, pH 7.0, recipe to follow:
50mM phosphate buffer:
1.08 g Sodium phosphate monobasic (Na2HPO4*H2O)
3.27 g Sodium phosphate dibasic (Na2HPO4*7 H2O)
q.s. to 200mL with milli-Q water
pH should be 7.0
1X PGAP buffer:
25 mL Phosphate buffer pH 7.0
39 mg DTT
9.3 mg EDTA
Dissolve lyophilized enzyme in 100 µl fresh 1X PGAP buffer.

Add 1 mU Pyrococcus furiosus Pyroglumate Aminopeptidase (TaKaRa)*. 10 µl equals 1 mU. Freeze the
remaining enzyme in 10 µl aliquots at -80oC. Incubate sample for 1 hours at 90oC.
Note: Watch reaction to be sure PVDF membrane does not dry out.
1. Discard enzyme solution. Rinse 3 times with 200 µl MQ water.
2. Transfer PVDF to new 0.5 ml eppendorf tube.
3. Dry in speedvac, and store in refrigerator @ 5 degree.

II. In Solution.
1. Lyophilize a few µg blocked protein. Use neurotensin or other known PyroGlu-containing protein as a control.
2. REDUCE. Add 100 µl 6M Guan-HCl, 0.2M Tris buffer titrated with glacial acetic acid to pH 8.0, 0.01M DTT (1.54 mg DTT in 1 ml of 6M Guan-HCl 0.2M Tris stock solution). Incubate at 45oC for 1 hour.
3. DESALT. Add protein solution to a Microcon-10 cartridge and spin at 14,000 g for 8 minutes.
4. WASH. Add 100 µl Milli-Q water and spin at 14,000 g until 10 µl remains, approximately 8 minutes. Invert and spin 2 minutes at 1000 g.
5. DIGEST. Add 40 µl of freshly made 1X PGAP buffer consisting of 50 mM Phosphate, 10 mM DTT, 1 mM EDTA, pH 7.0, recipe to follow.

50mM phosphate buffer:
1.08 g Sodium phosphate monobasic (Na2HPO4* H2O)
3.27 g Sodium phosphate dibasic (Na2HPO4*7 H2O)
q.s. to 200mL with milli-Q water
pH should be 7.0
1X PGAP buffer:
25 mL Phosphate buffer pH 7.0
39 mg DTT
9.3 mg EDTA
Dissolve lyophilized enzyme in 100 µl fresh 1X PGAP buffer. Add 1 mU enzyme (TaKaRa)*. 10 µl equals 1 mU. Freeze the remaining enzyme in 10 µl aliquots at -80oC. Incubate sample for 2 hours at 75oC.
6. DESALT. Repeat steps 3 & 4 until 10 µl remain, add 10 µl 2X Tris-Gly reduced sample buffer, invert and spin 2 minutes at 1000 g.
7. Load samples to a 4-20% TG gel, electroblot, and sequence.
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> Pyrococcus furiosus Pyroglutamate Aminopeptidase (TaKaRa TAK7334)
- Note: Stability of the enzyme:
- Lyophilized: room temp for 1 year 100% active
- Dissolved: -20oC for 1 month 100% active, 5oC for 1 month 70% active
> REAGENTS:
- Polyvinyl pyrrolidine (PVP-360) FW 360,000
- Tris (hydroxymethyl)aminomethane FW 121.14
- Ethylenediaminetetraacetic acid tetra sodium salt (EDTA)
- C10H12N2O8Na4*4 H2O FW 452.2
- Dithiothreitol (DTT) C4H10O2S2 FW 154.25
- Sodium phosphate monobasic Na2HPO4* H2O FW 120.0
- Sodium phosphate dibasic Na2HPO4*7 H2O FW 142.0

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